GnRH-induced Gonadotropin Secretion Research Articles

PREDICTIVE VALUE OF FOLLICULAR FLUID GHRELIN IN WOMEN WITH POLYCYSTIC OVARIAN DISEASE WHO UNDERGO INTRACYTOPLASMIC SPERM INJECTION

Ghrelin’s role in reproduction was first suggested by its wide expression in many human reproductive tissues including its immunohistochemical expression in the human ovary. Ghrelin expression is identified in the seminiferous tubuli of the interstitial Leyding and Sertoli cells in male testicles, whereas it is identified in the ovarian hilus interstitial cells and mature corpus lutea in females.It is postulated that ghrelin plays a local regulatory role in the male and female gonads. In females, Ghrelin acts centrally by suppressing hypothalamic GnRH release and GnRH-induced gonadotropin secretion by the pituitary; and in gonads it acts by increasing luteolytic factors such as PGF2 alfa, and ghrelin inhibiting estradiol and progesterone biosynthesis in granulose-lutein cells as well as granulosa cell (GC) proliferation.This potential regulatory effect on steroidogenesis and ovarian cellular function led to the hypothesis that ghrelin may mediate the relationship between metabolism and oocyte quality in addition to possible role in the regulation of embryo development in as the cleavage development before the six-cell stage of a preimplantation human embryo depends solely on maternally inherited material. Ghrelin levels can, therefore, be an indicator of an underlying pathology as in humans in states of chronic malnutrition such as anorexia nervosa, serum ghrelin levels may be increased, while in other conditions it could be associated with decreased ghrelin levels such as obesity and polycystic ovarian syndrome (PCOS).

Administration of Very Low Doses of Estradiol Modulates the LH Response to a GnRH Bolus and the LH and Cortisol Responses to Naloxone Infusion in Patients with Functional Hypothalamic Amenorrhea (FHA): A Pilot Study

Background: Functional Hypothalamic Amenorrhea (FHA) is a stress-induced blockade of the reproductive axis. Such impairment is mainly due to altered control of GnRH-induced gonadotropin secretion as well as alterations of other endocrine functions. Methods: Seventeen patients with FHA participated in the study. Basal hormonal profiles and GnRH and Naloxone tests for LH (Luteinizing Hormone) and for LH and cortisol responses, respectively, were performed before and after two weeks of administration of a very low dose of estradiol (2.5 ng two times a day). Results: The treatment improved both gonadotropins, mainly LH. The LH response to the GnRH test improved in terms of the peak amplitude, as evaluated using Instantaneous Secretory Rates (ISR) computation. Moreover, when performing the Naloxone test after the treatment interval, FHA patients showed a quicker LH response and recovery of the cortisol response. Conclusions: Our study supports the relevance of very low dose estradiol priming to promote and restore impaired neuroendocrine function in patients with FHA.

Modulatory effects of l-carnitine plus l-acetyl-carnitine on neuroendocrine control of hypothalamic functions in functional hypothalamic amenorrhea (FHA)

Functional hypothalamic amenorrhea (FHA) is a relatively frequent disease due to the combination of metabolic, physical, or psychological stressors. It is characterized by the low endogenous GnRH-induced gonadotropin secretion, thus triggering the ovarian blockade and a hypoestrogenic condition. Up to now various therapeutical strategies have been proposed, both using hormonal treatment as well as neuroactive compounds. Since carnitine, namely l-acetyl-carnitine (LAC), has been demonstrated to be effective in the modulation of the central hypothalamic control of GnRH secretion, we aimed to evaluate whether a combined integrative treatment for 12 weeks of LAC (250 mg/die) and l-carnitine (500 mg/die) was effective in improving the endocrine and metabolic pathways in a group of patients (n = 27) with FHA. After the treatment, interval mean LH plasma levels increased while those of cortisol and amylase decreased significantly. When patients were subdivided according to baseline LH levels, only hypo-LH patients showed the significant increase of LH plasma levels and the significant decrease of both cortisol and amylase plasma levels. The increased 17OHP/cortisol ratio, as index of the adrenal activity, demonstrated the reduced stress-induced adrenal activity. In conclusion, our data sustain the hypothesis that the integrative administration of LAC plus l-carnitine reduced both the metabolic and the neuroendocrine impairment of patients with FHA.

Long-term GnRH-induced gonadotropin secretion in a novel hypothalamo-pituitary slice culture from tilapia brain

Organotypic cultures, prepared from hypothalamo-pituitary slices of tilapia, were developed to enable long-term study of secretory cells in the pituitary of a teleost. Values of membrane potential at rest were similar to those recorded from acute slices, and cells presented similar spontaneous spikes and spikelets. Some cells also exhibited slow spontaneous oscillations in membrane potential, which may be network-driven. Long-term (6days) continuous exposure to GnRH induced increases in LH and FSH secretion. FSH levels reached the highest levels after 24h of exposure to GnRH, and the highest secretion of LH was observed in days 4 and 5 of the experiment. Since slices were viable for several weeks in culture, maintaining the original cytoarchitecture, electrical membrane properties and the ability to secrete hormones in response to exogenous GnRH, this technique is ideal for studying the mechanisms regulating cell-to-cell communication under conditions resembling the in vivo tissue organization.

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E₂) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E₂, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E₂- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E₂, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.

Kisspeptin and KISS1R: a critical pathway in the reproductive system

In 2003, three groups around the world simultaneously discovered that KISS1R (GPR54) is a key gatekeeper of sexual maturation in both mice and men. Developmental changes in the expression of the ligand for KISS1R, kisspeptin, support its critical role in the pubertal transition. In addition, kisspeptin, a powerful stimulus of GNRH-induced gonadotropin secretion and may modulate both positive and negative sex steroid feedback effects at the hypothalamic level. Genetic studies in humans have revealed both loss-of-function and gainof-function mutations in patients with idiopathic hypogonadotropic hypogonadism and precocious puberty respectively. This review examines the kisspeptin/KISS1R pathway in the reproductive system.

Oxytocin is not important for the control of gonadotrophin secretion in the late follicular phase of the cycle

To study the role of oxytocin in basal and GnRH-induced gonadotrophin secretion in normal women. Normal women were studied in three cycles. When the diameter of the leading follicle was 15-16 mm, the women were infused intravenously (i.v.) for 3 h with normal saline (cycle 1), atosiban (cycle 2) or oxytocin (cycle 3). The study included 12 normally cycling women aged 23-38 years. After cessation of treatment, two injections of GnRH, 10 microg each, were administered i.v. 2 h apart and blood samples were collected every 30 min for a total of 240 min. The 30-min pituitary response (sensitivity) to a single GnRH injection (10 microg i.v.) was investigated thereafter every 12 h from the end of the 3-h infusion until the day of LH surge onset. No significant differences in LH and FSH response to GnRH (sensitivity and reserve) during the 240-min experiment were found between the three cycles. The time of LH surge onset from the initiation of the infusion was similar in the three cycles. Also similar in the three cycles were oestradiol (E2) and gonadotrophin levels as well as the 30-min response to GnRH for 48 h following the 3-h infusion. The present study demonstrates that neither exogenous oxytocin administration nor blockage of endogenous oxytocin action influences pituitary sensitivity to GnRH in cycling women.

Effects of Ghrelin upon Gonadotropin-Releasing Hormone and Gonadotropin Secretion in Adult Female Rats: In vivo and in vitro Studies

A reproductive facet of ghrelin, a stomach-derived orexigenic peptide involved in energy homeostasis, has been recently suggested, and predominantly inhibitory effects of ghrelin upon luteinizing hormone (LH) secretion have been demonstrated in rat models. Yet, the modulatory actions of ghrelin on the gonadotropic axis remain scarcely evaluated. We report herein a detailed analysis of the effects of ghrelin upon LH and follicle-stimulating hormone (FSH) secretion in the female rat, using a combination of in vivo and in vitro approaches. Intracerebroventricular administration of ghrelin (3 nmol/rat) evoked a significant inhibition of LH secretion in cyclic female rats throughout the estrous cycle (proestrus afternoon, estrus, metestrus), as well as in ovariectomized females. In good agreement, gonadotropin-releasing hormone (GnRH) secretion by hypothalamic fragments from ovariectomized females was significantly inhibited by ghrelin. In contrast, ghrelin dose-dependently stimulated basal LH and FSH secretion by pituitary tissue in vitro; a phenomenon that was proven dependent on the phase of estrous cycle, as it was neither detected at estrus nor observed after ovariectomy. Conversely, GnRH-stimulated LH secretion in vitro was persistently inhibited by ghrelin regardless of the stage of the cycle, whereas stimulated FSH secretion was only inhibited by ghrelin at estrus. In addition, cyclic fluctuations in mRNA levels of growth hormone secretagogue receptor (GHS-R)1a, i.e. the functional ghrelin receptor, were observed in the pituitary, with low values at estrus and metestrus. GHS-R1a mRNA levels, however, remained unchanged after ovariectomy. In summary, our data illustrate a complex mode of action of ghrelin upon the gonadotropic axis, with predominant inhibitory effects at central (hypothalamic) levels and upon GnRH-induced gonadotropin secretion, but direct stimulatory actions on basal LH and FSH secretion. Overall, our results further document the reproductive role of ghrelin, which might be relevant for the integrated control of energy balance and reproduction.

Effects of clomiphene and raloxifene on gonadotrophin secretion in postmenopausal women: evidence of nongenomic action.

To evaluate the effect of raloxifene (R) and clomiphene (Cl) on FSH and LH secretion in postmenopausal women. Postmenopausal volunteer women participated in two experimental (Exp) procedures. In Group 1, the women received R (180 mg/day orally) for 30 days plus oestradiol (E2) through skin patches (100 microg/24 h) from days 21 to 30 (R-Exp). After a month's break the same women received Cl (150 mg/day orally) for 30 days plus E2 as above (Cl-Exp). In Group 2, the women received E2 for 30 days plus R from days 21 to 30 (R-Exp) and after a month's break they received E2 for 30 days plus Cl from days 21 to 30 (Cl-Exp). Daily doses were as in Group 1. A GnRH test (100 microg intravenously) was performed in all women on days 0, 10, 20 and 30 of each experiment. Sixteen healthy postmenopausal women were divided into two groups (eight women in each group). The area under the curve (AUC) of DeltaFSH and DeltaLH response to GnRH (net increase above the basal value) was calculated. In Group 1, basal levels of FSH and LH did not change significantly during the R-Exp, while they decreased significantly in the Cl-Exp (P < 0.001). The addition of E2 did not have any effect. The AUC of LH response to GnRH increased significantly in the R-Exp (P < 0.05) and that of FSH in the Cl-Exp (P < 0.05). In Group 2, basal levels of FSH and LH declined significantly during treatment with E2 in both the R-Exp (P < 0.01) and the Cl-Exp (P < 0.001). However, the addition of Cl (for 10 days) interrupted this decrease, while the addition of R stimulated FSH levels significantly (P < 0.05). E2 suppressed significantly the AUC of LH in both experiments (P < 0.05). The addition of Cl did not affect the AUC in response to GnRH, while the addition of R increased the AUC of both LH and FSH (P < 0.05). These results demonstrate for the first time that in contrast to Cl, R does not exert oestrogenic effects on basal gonadotrophin secretion. Although the antioestrogenic action of these drugs was evident only after pretreatment with E2, both R and Cl stimulated GnRH-induced gonadotrophin secretion in oestrogen-deprived women. It is hypothesized that these two compounds sensitize the pituitary to GnRH through mechanisms not involving the oestrogen receptor complex (nongenomic).

FSH and LH responses to GnRH after ovariectomy in postmenopausal women.

Whether the postmenopausal ovary is still playing a role in the control of gonadotrophin secretion in response to GnRH has not been investigated. The aim of the present study was to test this hypothesis by examining changes in basal and GnRH-induced gonadotrophin secretion in postmenopausal women after bilateral ovariectomy. The responses of LH and FSH to GnRH [10 microg intravenously (i.v.)] were investigated in postmenopausal women from 2 days before to 8 days after total abdominal hysterectomy plus bilateral ovariectomy. Nine postmenopausal women aged 52-67 years and between 5 and 15 years after menopause. In all cases the ovaries were histologically normal. Pituitary responses to GnRH were calculated every 12-24 h as the net increases in LH (DeltaLH) and FSH (DeltaFSH) at 30 min above the basal values. Basal values of oestradiol (E2) and testosterone were also measured. Basal values of FSH showed a significant decrease on postoperative days 2 (P < 0.01) and 8 (P = 0.03) as compared to day 0, while at the same time points after the operation LH values were marginally lower than on day 2 (P = 0.05). Serum E2 values showed a gradual increase up to postoperative day 1 (P = 0.04) and a gradual decline thereafter. Basal testosterone concentrations decreased gradually and significantly after ovariectomy and were significantly lower on day 8 than on day 0 (P < 0.01). DeltaFSH and DeltaLH responses to GnRH did not change significantly with time. A temporary increase at 12 h after the operation was not significant. These results demonstrate for the first time that the removal of the ovaries in postmenopausal women does not affect GnRH-induced gonadotrophin secretion in the short term. It is suggested that the postmenopausal ovary is not a dominant regulator of hypothalamic-pituitary interactions.

Evidence of differential control of FSH and LH responses to GnRH by ovarian steroids in the luteal phase of the cycle.

It is known that during the follicular phase of the cycle, estradiol sensitizes the pituitary to GnRH. The aim of this study was to determine the role of ovarian steroids in the control of GnRH-induced gonadotrophin secretion in the luteal phase of the cycle. Eighteen normally cycling women were studied during the week following bilateral ovariectomy plus hysterectomy performed in early to mid-luteal phase. Six of the women received no hormonal treatment post-operatively (group 1, control), six received estradiol through skin patches (group 2) and the remaining six received estradiol plus progesterone (group 3). In all women the response at 30 min of LH (deltadeltaLH) and FSH (deltadeltaFSH) to GnRH (10 microg i.v.) was investigated on a daily basis. In group 1, serum FSH, LH and deltadeltaFSH values increased progressively following ovariectomy, while in groups 2 and 3 this increase was postponed or abolished. In contrast to deltadeltaFSH, deltadeltaLH values showed the same pattern of changes in all three groups with a significant decline up to post-operative day 4 and a gradual increase thereafter. These results demonstrate, for the first time, that in the early to mid-luteal phase of the cycle, estradiol and progesterone participate in the control of GnRH-induced FSH, but not LH, secretion. It is possible that in the luteal phase, the response of LH to GnRH is partly regulated by gonadotrophin surge attenuating factor.

In vitro nitric oxide effects on basal and gonadotropin-releasing hormone-induced gonadotropin secretion by pituitary gland of male crested newt (Triturus carnifex) during the annual reproductive cycle.

The objective of this study was to test the possible nitric oxide (NO) involvement in pituitary gonadotropin secretion in the male crested newt, Triturus carnifex. Pituitaries were incubated in vitro with medium alone, GnRH, NO donor (NOd, sodium nitroprusside), NO synthase inhibitor (NOSi, Nomega-nitro-L-arginine methyl ester), cGMP analogue (cGMPa, 8-bromo-cGMP), soluble guanylate cyclase inhibitor (sGCi, cystamine), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi during the annual reproductive cycle: pre-reproduction, reproduction (noncourtship and courtship), and the refractory, recovery, and estivation periods. To determine pituitary gonadotropin secretion indirectly, newt testes were superfused in vitro with preincubated pituitaries, and androgen release was determined. NO synthase (NOS) activity and cGMP levels were assessed in the preincubated pituitaries. Medium alone- and GnRH-preincubated pituitary increased androgen secretion during pre-reproduction, noncourtship, courtship, and recovery; the GnRH-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOd and cGMPa increased androgens in all reproductive phases considered except courtship; the NOd- and cGMP-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOS activity was highest during courtship and lowest during the refractory and estivation periods. GnRH increased NOS activity during pre-reproduction, noncourtship, and recovery. Cyclic GMP levels were highest during courtship and lowest during the refractory period and estivation. GnRH increased cGMP levels during pre-reproduction, noncourtship, and recovery, while NOd did so during all reproductive phases considered. These results suggest that basal and GnRH-induced gonadotropin secretion are up-regulated by NO in the pituitary gland of the male Triturus carnifex.

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.

Nitric oxide mediates gonadotropin-releasing hormone effects on frog pituitary.

We studied the possible role of nitric oxide (NO) in GnRH-induced gonadotropin secretion in the female water frog, Rana esculenta. During pre-reproduction, pre-ovulation, ovulation, post-ovulation, refractory, recovery and hibernation, pituitaries were incubated with medium-alone, GnRH, NO donor (NOd), NO synthase inhibitor (NOSi), cyclic GMP analogue (cGMPa), soluble guanylate cyclase inhibitor (sGCi), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi. Because antisera raised against gonadotropins are not available for this species, we measured these hormones indirectly through their effects on ovarian progesterone secretion. The ovaries were superfused with the pituitaries pre-incubated as reported above. In addition, NOS activity and cGMP levels were determined in the pre-incubated pituitaries. Those pre-incubated with medium-alone and with GnRH increased progesterone secretion during pre-reproduction, pre-ovulation, ovulation and recovery; the increase induced by GnRH was higher than that induced by medium-alone during pre-reproduction, pre-ovulation and recovery. NOd and cGMPa increased progesterone in all considered reproductive phases except ovulation; the increase induced by NOd and cGMP was higher than that induced by medium-alone during pre-reproduction, pre-ovulation and recovery. NOS activity was highest during ovulation and lowest during post-ovulation, refractory and hibernation. GnRH increased NOS activity during pre-reproduction, pre-ovulation and recovery. Cyclic GMP levels were highest during ovulation and lowest during post-ovulation, refractory and hibernation. GnRH increased cGMP levels during pre-reproduction, pre-ovulation and recovery, NOd during all considered reproductive phases. These results suggest that NO mediates basal and GnRH-induced gonadotropin secretion in female Rana esculenta.

Absence of Photoperiodic Modulation of Gonadotrophin Secretion in HPD Rams Following Chronic Pulsatile Infusion of GnRH

Hypothalamo-pituitary disconnected Soay rams (HPD rams) were treated chronically with a pulsatile infusion of GnRH (250 ng/2 h) for 10 weeks to reactivate the pituitary-gonadal axis under long days (16L:8D; LD group n=8) and short days (8L:16D, SD group n=8). The aim was to establish whether photoperiod modulates the secretion of gonadotrophins (presumably through the daily melatonin signal) by an action at the level of the pituitary gland. The treatment of the HPD rams with GnRH induced a marked, long-term increase in the peripheral blood concentrations of FSH, LH and testosterone, growth of the testes and reappearance of the androgen-dependent sexual skin colouration. There were parallel changes in the short-term, pulsatile fluctuations in the blood concentrations of LH and FSH, the pituitary responsiveness to GnRH and the testicular responsiveness to a standardized injection of oLH, as observed previously in normal intact rams during reactivation of the reproductive axis induced by a switch from long to short days. There were no significant differences in any of the reproductive parameters between the LD and SD HPD groups, although the blood concentrations of prolactin were significantly higher in the LD group, and GnRH provoked a chronic increase in the blood concentrations of prolactin, particularly in the SD group. Overall, the results support the conclusion that (i) melatonin does not act within the pituitary gland to mediate effects of photoperiod on GnRH-induced gonadotrophin secretion; and (ii) changes in prolactin secretion which result from the local action of melatonin in the pituitary gland do not affect gonadotrophin secretion in the ram.

Expression of ryanodine receptors in the pituitary gland: evidence for a role in gonadotropin-releasing hormone signaling.

GnRH elicits secretion of LH and FSH from gonadotropes by activating an array of intracellular signals including the generation of inositol triphosphate and the release of intracellular calcium. Given the important role of calcium in the secretory responses to GnRH, we examined the expression and function of the ryanodine receptors, which are known to modulate calcium release from intracellular stores. Using RT-PCR analysis, we found that ryanodine receptor (RyR) types 2 and 3, but not type 1, are expressed in rat pituitaries. Pulses of GnRH were administered to perifused primary rat pituitary cells in the presence or absence of a ryanodine receptor antagonist, ruthenium red, to assess effects on GnRH-mediated LH secretion. Treatment with ruthenium red resulted in a 40% decrease in the spike phase of GnRH-induced LH release and a 35% reduction in the plateau phase. Ruthenium red also inhibited GnRH-mediated transcription of a transfected alpha-LUC reporter plasmid. RyR messenger RNA (mRNA) expression varied during the rat estrous cycle with maximal levels following increases of progesterone. The effects of gonadal steroids on pituitary RyR mRNA levels were examined directly in ovariectomized rats that were treated with estrogen (E), or estrogen and progesterone (P). In this paradigm, E decreased, whereas E + P increased RyR3 mRNA levels. These results indicate that RyR is expressed and hormonally regulated in the rat pituitary and suggest that it might play a role in mediating GnRH-induced gonadotropin synthesis and secretion.

Expression of Ryanodine Receptors in the Pituitary Gland: Evidence for a Role in Gonadotropin-Releasing Hormone Signaling

GnRH elicits secretion of LH and FSH from gonadotropes by activating an array of intracellular signals including the generation of inositol triphosphate and the release of intracellular calcium. Given the important role of calcium in the secretory responses to GnRH, we examined the expression and function of the ryanodine receptors, which are known to modulate calcium release from intracellular stores. Using RT-PCR analysis, we found that ryanodine receptor (RyR) types 2 and 3, but not type 1, are expressed in rat pituitaries. Pulses of GnRH were administered to perifused primary rat pituitary cells in the presence or absence of a ryanodine receptor antagonist, ruthenium red, to assess effects on GnRH-mediated LH secretion. Treatment with ruthenium red resulted in a 40% decrease in the spike phase of GnRH-induced LH release and a 35% reduction in the plateau phase. Ruthenium red also inhibited GnRH-mediated transcription of a transfected α-LUC reporter plasmid. RyR messenger RNA (mRNA) expression varied during the rat estrous cycle with maximal levels following increases of progesterone. The effects of gonadal steroids on pituitary RyR mRNA levels were examined directly in ovariectomized rats that were treated with estrogen (E), or estrogen and progesterone (P). In this paradigm, E decreased, whereas E + P increased RyR3 mRNA levels. These results indicate that RyR is expressed and hormonally regulated in the rat pituitary and suggest that it might play a role in mediating GnRH-induced gonadotropin synthesis and secretion.

Ovarian steroids modulate endothelin-induced luteinizing hormone secretion from cultured rat pituitary cells.

It has been demonstrated that endothelins (ETs) induce LH secretory responses in cultured rat pituitary cells. Because estradiol and progesterone are known to be potent modulators of GnRH-induced gonadotropin secretion, we examined whether these steroids also influence the secretory responses of gonadotrophs to ETs. Cultured female rat pituitary cells were treated for 48 h with vehicle (0.2% ethanol), 1 nM estradiol alone, or a combination of 1 nM estradiol and 100 nM progesterone or for 48 h with 1 nM estradiol and a further 4 h with 100 nM progesterone and subsequently stimulated with 10 pM-100 nM ET-1 or ET-3. Forty-eight-hour estradiol treatment led to enhanced LH secretory responses to both ETs. This action was facilitated by short term progesterone treatment (3-fold vs. vehicle), while long term progesterone treatment was inhibitory. Perifusion experiments were performed to study the kinetics of individual and pulsatile LH secretory responses after steroid exposure of pituitary cells. ET-1 induced immediate biphasic LH responses that were augmented by long term estradiol treatment. Two-hour progesterone exposure led to marked increases in LH secretion, whereas 48-h progesterone treatment was inhibitory. Estradiol and progesterone were able to modulate both the initial spike and the secondary plateau phase of the secretory profile in response to ET-1, although these actions did not always reach statistical significance. The steroid treatment paradigms employed also induced inhibitory and stimulatory effects in cells that were stimulated with ET-3 in a pulsatile fashion. In these experiments it could be demonstrated that the facilitatory action of progesterone was present after 50 min of treatment and was maximum after 150 min (5-fold enhancement). The present data support the hypothesis that ETs are involved in the physiological regulation of gonadotropin secretion and demonstrate that ovarian steroids can act as potent modulators of ET-induced LH secretion.

Hormonal characteristics of free-ranging female lions (Panthera leo) of the Serengeti Plains and Ngorongoro Crater

Pituitary responses to gonadotrophin-releasing hormone (GnRH) and prolactin and steroid secretory profiles were examined in two populations of adult, female lions in the Serengeti (one outbred in the Serengeti Plains and one inbred in the Ngorongoro Crater) to determine whether reductions in genetic variability adversely affected endocrine function. GnRH-induced gonadotrophin secretion was also examined after adrenocorticotrophic hormone (ACTH) treatment to determine whether acute increases in serum cortisol altered pituitary function. Anaesthetized lions were administered (i) saline i.v. after 10 and 100 min of blood sampling, (ii) saline at 10 min and GnRH (1 micrograms kg-1 body weight) after 100 min; or (iii) ACTH (3 micrograms kg-1) at 10 min and GnRH after 100 min of sampling. Basal serum cortisol and basal and GnRH-induced gonadotrophin secretion were similar (P > 0.05) between females of the Ngorongoro Crater and Serengeti Plains. After ACTH, serum cortisol increased two- to threefold over baseline values and the response was unaffected (P > 0.05) by location. ACTH-induced increases in serum cortisol had no effect on subsequent basal or GnRH-stimulated luteinizing hormone (LH) or follicle-stimulating hormone (FSH) secretion. Overall mean serum progesterone concentrations ranged from 0.2 to 5.4 ng ml-1 with the exception of four females (two in the Serengeti and two in the Crater; progesterone range, 18.4-46.5 ng ml-1) that were presumed pregnant (three of these females were observed nursing cubs several weeks later).(ABSTRACT TRUNCATED AT 250 WORDS)